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• W2005444914 abstract "RGS proteins are defined by the presence of a semiconserved RGS domain that confers the GTPase-activating activity of these proteins toward certain Gα subunits. RGS6 is a member of a subfamily of RGS proteins distinguished by the presence of DEP and GGL domains, the latter a Gβ5-interacting domain. Here we report identification of 36 distinct transcripts of human RGS6 that arise by unusually complex processing of the RGS6 gene, which spans 630 kilobase pairs of genomic DNA in human chromosome 14 and is interrupted by 19 introns. These transcripts arise by use of two alternative transcription sites and complex alternative splicing mechanisms and encode proteins with long or short N-terminal domains, complete or incomplete GGL domains, 7 distinct C-terminal domains and a common internal domain where the RGS domain is found. The role of structural diversity in the N-terminal and GGL domains of RGS6 splice variants in their interaction with Gβ5 and subcellular localization and of Gβ5 on RGS6 protein localization was examined in COS-7 cells expressing various RGS6 splice variant proteins. RGS6 splice variants with complete GGL domains interacted with Gβ5, irrespective of the type of N-terminal domain, while those lacking a complete GGL domain did not. RGS6 protein variants displayed subcellular distribution patterns ranging from an exclusive cytoplasmic to exclusive nuclear/nucleolar localization, and co-expression of Gβ5 promoted nuclear localization of RGS6 proteins. Analysis of our results show that the long N-terminal and GGL domain sequences of RGS6 proteins function as cytoplasmic retention sequences to prevent their nuclear/nucleolar accumulation. These findings provide the first evidence for Gβ5-independent functions of the GGL domain and for a role of Gβ5 in RGS protein localization. This study reveals extraordinary complexity in processing of the human RGS6 gene and provides new insights into how structural diversity in the RGS6 protein family is involved in their localization and likely function(s) in cells. RGS proteins are defined by the presence of a semiconserved RGS domain that confers the GTPase-activating activity of these proteins toward certain Gα subunits. RGS6 is a member of a subfamily of RGS proteins distinguished by the presence of DEP and GGL domains, the latter a Gβ5-interacting domain. Here we report identification of 36 distinct transcripts of human RGS6 that arise by unusually complex processing of the RGS6 gene, which spans 630 kilobase pairs of genomic DNA in human chromosome 14 and is interrupted by 19 introns. These transcripts arise by use of two alternative transcription sites and complex alternative splicing mechanisms and encode proteins with long or short N-terminal domains, complete or incomplete GGL domains, 7 distinct C-terminal domains and a common internal domain where the RGS domain is found. The role of structural diversity in the N-terminal and GGL domains of RGS6 splice variants in their interaction with Gβ5 and subcellular localization and of Gβ5 on RGS6 protein localization was examined in COS-7 cells expressing various RGS6 splice variant proteins. RGS6 splice variants with complete GGL domains interacted with Gβ5, irrespective of the type of N-terminal domain, while those lacking a complete GGL domain did not. RGS6 protein variants displayed subcellular distribution patterns ranging from an exclusive cytoplasmic to exclusive nuclear/nucleolar localization, and co-expression of Gβ5 promoted nuclear localization of RGS6 proteins. Analysis of our results show that the long N-terminal and GGL domain sequences of RGS6 proteins function as cytoplasmic retention sequences to prevent their nuclear/nucleolar accumulation. These findings provide the first evidence for Gβ5-independent functions of the GGL domain and for a role of Gβ5 in RGS protein localization. This study reveals extraordinary complexity in processing of the human RGS6 gene and provides new insights into how structural diversity in the RGS6 protein family is involved in their localization and likely function(s) in cells. RGS 1The abbreviations used are: RGS, regulators of G protein signaling; bp, base pair(s); DEP, disheveled, Egl-10, pleckstrin homology; DAB, 3,3′-diaminobenzidine; DMEM, Dulbecco's modified Eagle's medium; DPBS, Dulbecco's phosphate-buffered saline; GFP, green fluorescent protein; EGFP, enhanced GFP; FITC, fluorescein isothiocyanate; Gα, α-subunit of G protein, Gβ, β-subunit of protein; GAP, GTPase-activating protein; Gγ, G protein γ-subunit; G protein, guanine nucleotide-binding protein; GGL, G protein γ-subunit-like domain; NES, nuclear export sequence; NLS, nuclear localization sequence; NoLS, nucleolar localization sequence; RACE, rapid amplification of cDNA ends; RGD, RGS domain; PI, propidium iodide. proteins comprise a family of proteins, defined by the presence of a semiconserved region called the RGD, that have been implicated in the negative regulation of heterotrimeric G protein signaling (1Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 2De Vries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (513) Google Scholar, 3Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (937) Google Scholar). The existence of such proteins was first shown by genetic studies in yeast (4Dohlman H.G. Apaniesk D. Chen Y. Song J. Nusskern D. Mol. Cell. Biol. 1995; 15: 3635-3643Crossref PubMed Scopus (168) Google Scholar) and Caenorhabditis elegans (5Koelle M.R. Horvitz H.R. Cell. 1996; 84: 115-125Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar) where the yeast pheromone desensitization factor Sst2p and the C. elegans Sst2p homolog Egl-10 were found to negatively regulate Gpa1 and GOA-1, respectively, both homologs of the mammalian heterotrimeric G protein Gαo. The presence of a semiconserved domain of ∼120 amino acids in Sst2p and Egl-10 (i.e. the RGD) enabled Koelle and Horvitz (5Koelle M.R. Horvitz H.R. Cell. 1996; 84: 115-125Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar) to demonstrate the existence of a family of mammalian proteins with this domain. More than 20 mammalian genes encode proteins with this hallmark RGD or less related versions of this domain. Subsequent studies demonstrated that RGS proteins or their isolated RGDs display GTPase-activating protein activity toward Gi and Gq proteins (1Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 2De Vries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (513) Google Scholar, 3Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (937) Google Scholar), providing insight into how RGS proteins could function to turn off G proteins following their GTP-dependent activation by receptors. Some studies have suggested that RGS proteins may interact with the effectors adenylyl cyclase or phosphoinositide phospholipase C (6Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Crossref PubMed Scopus (338) Google Scholar, 7Sinnarajah S. Dessauer C.W. Srikumar D. Chen J. Yuen J. Yilma S. Dennis J.C. Morrison E.E. Vodyanoy V. Kehrl J.H. Nature. 2001; 409: 1051-1055Crossref PubMed Scopus (212) Google Scholar) or with receptors to block heterotrimeric G protein signaling (8Zeng W. Xu X. Popov S. Mukhopadhyay S. Chidiac P. Swistok J. Danho W. Yagaloff K.A. Fisher S.L. Ross E.M. Muallem S. Wilkie T.M. J. Biol. Chem. 1998; 273: 34687-34690Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Yet, the physiological function of RGS proteins has been shown only for pheromone signaling in yeast (4Dohlman H.G. Apaniesk D. Chen Y. Song J. Nusskern D. Mol. Cell. Biol. 1995; 15: 3635-3643Crossref PubMed Scopus (168) Google Scholar), neuronal signaling in C. elegans (5Koelle M.R. Horvitz H.R. Cell. 1996; 84: 115-125Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar), and, in mammals, phototransduction in the eye where RGS9 is required for the rapid inactivation of transducin (9He W. Cowan C.W. Wensel T.G. Neuron. 1998; 20: 95-102Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 10Chen C.K. Burns M.E. He W. Wensel T.G. Baylor D.A. Simon M.I. Nature. 2000; 403: 557-560Crossref PubMed Scopus (334) Google Scholar). Moreover, recent evidence from this and other laboratories has shown that several RGS proteins are localized predominantly at intracellular sites other than the plasma membrane including the nucleus (11Burgon P.G. Lee W.L. Nixon A.B. Peralta E.G. Casey P.J. J. Biol. Chem. 2001; 276: 32828-32834Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 12Chatterjee T.K. Fisher R.A. J. Biol. Chem. 2000; 275: 29660-29671Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 13Chatterjee T.K. Fisher R.A. J. Biol. Chem. 2000; 275: 24013-24021Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 14Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J.H. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar, 15Heximer S.P. Lim H. Bernard J.L. Blumer K.J. J. Biol. Chem. 2001; 276: 14195-14203Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 16Saitoh O. Masuho I. Terakawa I. Nomoto S. Asano T. Kubo Y. J. Biol. Chem. 2001; 276: 5052-5058Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), where G proteins as well as their effectors and activating receptors are not thought to localize. These findings raise the fascinating possibility that some members of the RGS protein family may have functions apart from, or in addition to, regulatory control of heterotrimeric G protein signaling. Five subfamilies of RGS proteins have been proposed based upon sequence identities within the RGD of these proteins (3Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (937) Google Scholar). Interestingly, this classification also groups proteins with similar structural domains outside of the RGD that may be involved in subcellular targeting, regulation, protein interaction(s) or specific functions of members of a given subfamily. Indeed, the R4 subfamily proteins RGS4 and RGS16 possess NESs that function to transport these proteins from the nucleus to the cytoplasm (13Chatterjee T.K. Fisher R.A. J. Biol. Chem. 2000; 275: 24013-24021Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) and the N-terminal domain of RGS3 has been implicated in its recruitment to the plasma membrane (14Dulin N.O. Sorokin A. Reed E. Elliott S. Kehrl J.H. Dunn M.J. Mol. Cell. Biol. 1999; 19: 714-723Crossref PubMed Google Scholar). RGS proteins in the RZ subfamily possess cysteine string motifs and several other protein domains have been identified that are unique to members of a given RGS protein subfamily (3Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (937) Google Scholar). Of particular interest to the present study is the R7 subfamily of RGS proteins that includes RGS6 as well as RGS7, RGS9, and RGS11. Each of these RGS proteins has an N-terminal domain that contains a DEP (disheveled, Egl-10 and pleckstrin homology) and a GGL (G-protein gamma subunit-like) domain. Although the function of the DEP domain is unknown, the GGL domain has been shown to represent a binding site for Gβ5 (17Snow B.E. Krumins A.M. Brothers G.M. Lee S.F. Wall M.A. Chung S. Mangion J. Arya S. Gilman A.G. Siderovski D.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13307-13312Crossref PubMed Scopus (231) Google Scholar, 18Snow B.E. Betts L. Mangion J. Sondek J. Siderovski D.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6489-6494Crossref PubMed Scopus (104) Google Scholar), an atypical Gβ subunit (19Watson A.J. Katz A. Simon M.I. J. Biol. Chem. 1994; 269: 22150-22156Abstract Full Text PDF PubMed Google Scholar). Present evidence suggests that R7 family members may represent physiological binding partners for Gβ5 (10Chen C.K. Burns M.E. He W. Wensel T.G. Baylor D.A. Simon M.I. Nature. 2000; 403: 557-560Crossref PubMed Scopus (334) Google Scholar, 20Witherow D.S. Wang Q. Levay K. Cabrera J.L. Chen J. Willars G.B. Slepak V.Z. J. Biol. Chem. 2000; 275: 24872-24880Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), rather than Gγ as observed for other Gβ proteins. The precise function of this interaction is not yet clear although Gβ5 binding to these RGS proteins has been implicated in RGS and Gβ5 protein stability (10Chen C.K. Burns M.E. He W. Wensel T.G. Baylor D.A. Simon M.I. Nature. 2000; 403: 557-560Crossref PubMed Scopus (334) Google Scholar, 18Snow B.E. Betts L. Mangion J. Sondek J. Siderovski D.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6489-6494Crossref PubMed Scopus (104) Google Scholar, 20Witherow D.S. Wang Q. Levay K. Cabrera J.L. Chen J. Willars G.B. Slepak V.Z. J. Biol. Chem. 2000; 275: 24872-24880Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), in regulation of RGS protein signaling or GAP activity (21Skiba N.P. Martemyanov K.A. Elfenbein A. Hopp J.A. Bohm A. Simonds W.F. Arshavsky V.Y. J. Biol. Chem. 2001; 276: 37365-37372Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 22He W. Lu L. Zhang X. El-Hodiri H.M. Chen C.K. Slep K.C. Simon M.I. Jamrich M. Wensel T.G. J. Biol. Chem. 2000; 275: 37093-37100Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 23Levay K. Cabrera J.L. Satpaev D.K. Slepak V.Z. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2503-2507Crossref PubMed Scopus (85) Google Scholar, 24Kovoor A. Chen C.K. He W. Wensel T.G. Simon M.I. Lester H.A. J. Biol. Chem. 2000; 275: 3397-3402Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 25Posner B.A. Gilman A.G. Harris B.A. J. Biol. Chem. 1999; 274: 31087-31093Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) and in localization of Gβ5 (26Zhang J.-H. Barr V.A. Mo Y. Rojkova A.M. Liu S. Simonds W.F. J. Biol. Chem. 2001; 276: 10284-10289Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). We undertook studies to clone members of the RGS protein family to further our understanding of the structural diversity and complexity within this family. Recent evidence suggests that this complexity may arise not only from the more than 20 mammalian genes encoding RGS protein members, but also by the existence of multiple forms of a given RGS gene product. We identified two major transcripts for human RGS3 (27Chatterjee T.K. Eapen A.K. Fisher R.A. J. Biol. Chem. 1997; 272: 15481-15487Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) and subsequently demonstrated the existence of twelve alternatively spliced forms of human RGS12 (12Chatterjee T.K. Fisher R.A. J. Biol. Chem. 2000; 275: 29660-29671Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). RGS9 has also been found to exist in two splice variant forms (28Granneman J.G. Zhai Y. Zhu Z. Bannon M.J. Burchett S.A. Schmidt C.J. Andrade R. Cooper J. Mol. Pharmacol. 1998; 54: 687-694PubMed Google Scholar). Here we report identification of 36 splice variant forms of RGS6 that arise by use of two alternative transcription start sites within the human RGS6 gene and by complex alternative splicing of the two primary RGS6 mRNAs. Identification of these transcripts enabled us to deduce the structure of the human RGS6 gene and the splicing mechanisms that generate these novel RGS6 transcripts. Interestingly, these RGS6 transcripts encode proteins with long or short N-terminal domains, complete or incomplete GGL domains, seven distinct C-terminal domains, and a common internal domain where the RGD is located. The existence of diversity outside of but not within the RGD of the RGS6 protein family raises complex questions in relation to a unifying hypothesis that these proteins have a single function dictated by this domain. We examined the role of structural diversity within the N-terminal and GGL domain of RGS6 splice variants in their interaction with Gβ5 and subcellular localization patterns and assessed whether Gβ5 alters the subcellular localization of RGS6 proteins. Our results demonstrate unique subcellular distribution patterns of RGS6 protein variants that can be ascribed to N-terminal and GGL domains of these proteins functioning as cytoplasmic retention sequences. The role of the GGL domain in cytoplasmic retention of RGS6 was observed in cells lacking Gβ5, providing the first evidence for a function of this domain independent of its interaction with Gβ5. Moreover, co-expression studies with Gβ5 and RGS6 proteins provide new evidence that Gβ5 interaction with RGS proteins promotes changes in their subcellular distribution. These results demonstrate extraordinary complexity in processing of the human RGS6 gene and provide new insight into how structural complexity in RGS6 proteins dictates their subcellular localization and possible functions. Materials—5′-RACE-ready cDNA, marathon-ready cDNA, Quickscreen cDNA library panel and pEGFP vector were purchased from Clontech. pCR2.1 and pCR3.1 were from Invitrogen. Elongase was from Invitrogen. Antibody to and cDNA encoding mouse Gβ5 was a generous gift of Dr. William Simonds (National Institutes of Health) and Dr. Vladen Slepak (University of Miami), respectively. Cell culture medium and serum was provided by the Diabetes Endocrinology Research Center (the University of Iowa). Oligonucleotide primers and other molecular biological reagents were obtained from the University of Iowa DNA Core. Polyclonal RGS6 antibodies were generated with a synthetic peptide immunogen corresponding to residues 1–19 of RGS6L by Biosynthesis Incorporated (Lewisville, TX). PCR Amplification of RGS6 cDNAs—Full-length cDNAs encoding various forms of RGS6 were amplified using a PCR-based strategy we described previously (12Chatterjee T.K. Fisher R.A. J. Biol. Chem. 2000; 275: 29660-29671Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). We utilized a 505-bp expressed sequence tag identified as RGS6 (GenBank™ accession number H09621) to design primers for use in 5′- and 3′-RACE to amplify overlapping segments of RGS6 cDNAs essentially as we described previously (12Chatterjee T.K. Fisher R.A. J. Biol. Chem. 2000; 275: 29660-29671Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Marathon-ready human brain cDNA (adapter sequences on both cDNA ends) were used as templates for 5′-RACE and 3′-RACE using adapter-specific forward or reverse primers in combination with appropriate RGS6 forward or reverse primers. This cDNA was synthesized from poly(A)containing mRNA. Resulting PCR products were cloned into pCR2.1, and sequence analysis of multiple clones revealed successful amplification of overlapping 5′- and 3′-cDNA fragments of RGS6 from brain cDNA. Sequence analysis of these clones revealed the existence of multiple splice variant forms of RGS6. cDNAs encoding these splice variant forms of RGS6 were amplified using forward and reverse primers encompassing the translational start and stop sites, respectively. cDNAs were cloned into pCR3.1 and double-stranded sequencing was performed by automated fluorescent dideoxynucleotide sequencing by the University of Iowa DNA Core Facility. Preparation of EGFP Constructs of RGS6 —Various RGS6 protein cDNAs were PCR-amplified using gene-specific primers incorporating restriction sites to facilitate their cloning into EGFP vector. First, amplified RGS6 protein cDNAs were cloned in the T/A cloning vector pCR2.1 (Invitrogen). Then, restriction enzyme digestion and agarose gel purification of the cloned RGS6 protein cDNAs was performed. RGS6 protein cDNAs were ligated to EGFP vector in-frame with its C-terminal or N-terminal EGFP sequence. Double-stranded sequencing of all cloned RGS6 protein cDNAs was performed by automated fluorescent dideoxynucleotide sequencing by the University of Iowa DNA Core Facility. Cell Culture and Transfection—COS-7 cells were grown in DMEM supplemented with 10% fetal bovine serum and gentamycin (50 μg/ml) (complete DMEM) in a 5% CO2 humidified atmosphere at 37 °C. COS-7 cells were transiently transfected with vectors containing various RGS6 protein cDNAs and mouse Gβ5 cDNA by electroporation using a BioRad Gene-Pulser. Typically, COS-7 cells (107/ml) were transfected with 40 μg of plasmid DNA at settings of 0.22 kV and 950 μF. Cells were diluted in complete DMEM and plated in two-chambered slides (Nunc) at a density of ∼106 cells/well. Transfected cells were used in experiments 40 h following transfection. Immunofluorescence and Immunohistochemistry—Cells were rinsed three times with DPBS before fixation for immunofluorescence. For visualization of GFP-tagged RGS6 proteins in COS-7 cells, cells were fixed by treatment with 4% paraformaldehyde for 20 min at room temperature followed by permeabilization with DPBS containing 0.1% Triton X-100 and 0.1% Nonidet P-40 for 10 min at room temperature. After permeabilization, cells were treated with DPBS containing 100 μg/ml RNase A (Roche Applied Science) for 20 min at room temperature prior to staining with propidium iodide. For immunocytochemical detection of expressed Gβ5 in COS-7 cells, cells were fixed and permeabilized by treatment with 50% methanol, 50% acetone for1hat4 °C prior to treatment with RNase A and incubation with anti-Gβ5 (∼1 μg/ml) in DPBS containing 5% bovine serum albumin for 1 h at room temperature. Cells then were rinsed three times with DPBS and incubated in FITC-conjugated secondary antibody (1 mg/ml) in DPBS for 20 min at room temperature. RNase A treatment and propidium iodide staining were performed as described above. Cells were air-dried and then mounted using Vecta Shield mounting solution. Confocal microscopy was performed as we described previously (13Chatterjee T.K. Fisher R.A. J. Biol. Chem. 2000; 275: 24013-24021Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Images shown are representative of a minimum of 1000 cells derived from four or more separate transfections. Immunohistochemistry was performed by Molecular Histology (MD) with synthetic peptide immunogen affinity-purified (SulfoLink, Pierce) anti-RGS6L. Briefly, tissue slides were incubated with and without anti-RGS6L, followed by biotinylated goat anti-rabbit antibody and strepatavidin-horseradish peroxidase prior to incubation with substrate (DAB). Hematoxylin counterstain (stains nuclei blue) was used. Co-immunoprecipitation of Gβ5 and RGS6 —For co-immunoprecipitation studies, COS-7 cells were co-transfected with RGS6-GFP and mouse Gβ5 cDNA and grown for 48 h in 6-well culture dishes. Cells were harvested by lysis with 1 ml of ice-cold RIPA buffer (150 mm NaCl, 50 mm Tris-HCl, pH 7.5, 0.5% deoxycholate, 1% Nonidet P-40, 6 mm MgCl2, 10 mm phenylmethylsulfonyl fluoride) followed by centrifugation at 16,000 × g for 1 min at 4 °C. Resulting supernatants were incubated with anti-GFP antibodies overnight at 4 °C, followed by addition of protein A-Sepharose and incubation for an additional 3 h. Immunoprecipitates were collected by centrifugation and washed three times in RIPA buffer, suspended in Laemmli sample buffer and boiled for 3 min. Proteins were subjected to SDS-PAGE and immunoblotting as described previously (12Chatterjee T.K. Fisher R.A. J. Biol. Chem. 2000; 275: 29660-29671Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Identification of Human RGS6 cDNAs—We used a PCR-based strategy to first amplify and clone two RGS6 cDNAs (Genbank™ AF073920, AF073921), the first identified RGS6 cDNAs, using sequence information from a 505-bp expressed sequence tag (GenBank™ H09621). The naming of these cDNAs was derived from the conceptual translation of rat brain PCR products of Koelle and Horvitz (5Koelle M.R. Horvitz H.R. Cell. 1996; 84: 115-125Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar) in which degenerate primers were used to amplify a semiconserved region, now known as the RGS domain, of a family of mammalian proteins sharing sequence identify with EGL-10, the RGS protein in C. elegans. Inspection of the sequence of these two RGS6 cDNAs suggested the possibility of splicing of RGS6 transcripts at their 5′-ends; the larger form encoded a protein with an additional 139 N-terminal amino acids, and the cDNAs had different 5′-untranslated sequences. An apparent difference at the 3′-ends of these two cDNAs was subsequently resolved by identifying a 2-bp sequencing error in the longer form. Thus, the two RGS6 cDNAs appeared to represent long and short N-terminal forms of RGS6 so the short form was named an RGS6 variant to highlight this possibility. Using 5′- and 3′-RACE and sequence information from these two RGS6 cDNAs, we were able to identify the existence of 36 novel RGS6 transcripts from oligo(dT)-primed cDNA from human brain. Sequence analysis of full or partial RGS6 cDNAs revealed that these variant RGS6 forms, although highly homologous, exhibit differences in their 5′-, 3′-, and internal sequences. Two different 5′-cDNA ends were identified that correspond to proteins with the long or short N-terminal sequences mentioned above. RGS6 cDNAs with each type of 5′-end existed in combination with seven different types of 3′-ends, encoding seven different C-terminal domains of the proteins. In addition, each of the RGS6 transcripts distinguished by different 5′- and 3′-ends existed in two forms that differed in the region encoding the G protein γ-subunit-like (GGL) domain that defines the subfamily of RGS proteins that includes RGS6, RGS7, RGS9, and RGS11. Sequence differences in this region encode RGS6 proteins with complete or incomplete GGL domains. The coding sequences of the 36 different RGS6 cDNAs encode proteins ranging from 284 to 490 amino acids. Fig. 1 illustrates the structure and naming of RGS6 proteins encoded by the 36 distinct RGS6 cDNAs identified here. We devised a nomenclature based upon the differing N- and C-terminal sequences (Table I) and the presence or absence of a complete GGL domain of the encoded RGS6 proteins. Long and short N-terminal forms of RGS6 were designated as RGS6L and RGS6S, respectively, and the C-terminal forms were designated α, β, γ, δ, ϵ, η, and ζ. RGS6L proteins have a 139 N-terminal sequence not present in the otherwise homologous RGS6S proteins. Likewise the forms lacking a 37 amino acid sequence that includes the C-terminal 25 amino acids of the GGL domain, designated (-GGL), are otherwise homologous to RGS6 proteins with complete GGL domains. Moreover, α and β forms exist in two forms, with (α1, β1) or without (α2, β2) an 18 amino acid sequence located N-terminal to their common C-terminal sequence (Table I). Thus, using this nomenclature, long N-terminal forms of RGS6 with the α1 terminus with and without the GGL domain are designated RGS6Lα1 and RGS6Lα1(-GGL), respectively. Fig. 1 also illustrates that all forms of RGS6 have an RGS domain and that RGS6S forms lack the DEP domain, whose function is presently unknown, present in RGS6L proteins.Table IDifferent C-terminal ends and exon 18 variants of RGS6 proteinNamePESEQGRRTSLEKFTRSVC Terminusα1+GKSLAGKRLTGLMQSSα2-GKSLAGKRLTGLMQSSβ1+LYSNTPLAKRPβ2-LYSNTPLAKRPγ+CLQLLFδ+LLFϵ+Gη-VWLLζ-KVSKVELP Open table in a new tab Structure of the Human RGS6 Gene—The finding that the identified RGS6 transcripts encode highly homologous proteins that differ only by the presence or absence of sequence cassettes or in their C-terminal sequences suggested these transcripts might arise by alternative splicing mechanisms. Using BLAST nucleotide sequence analysis of identified RGS6 cDNAs with human gene data banks we were able to deduce the complete structure of the human RGS6 gene. The RGS6 gene spans 629,635 bp of DNA of human chromosome 14 and is interrupted by 19 introns, and the 20 RGS6 gene exons range in size from 51 to >332 bp. The intron-exon organization of the hRGS6 gene in relation to RGS6Lα1 mRNA is shown in Fig. 2. Table II shows the sizes of introns and the intron-exon splice junction sequences in RGS6 transcripts. As shown, introns vary in size from 155 bp to 387 kb, and all of the splice acceptor and donor sequences agree with the GT/AG consensus sequence (29Padgett R.A. Grabowski P.J. Konarska M.M. Seiler S. Sharp P.A. Annu. Rev. Biochem. 1986; 55: 1119-1150Crossref PubMed Google Scholar). The RGS6 gene intron phasing is type 0 (the intron occurs between codons) for introns 2, 5, 7, 9–11, 15–19; type 1 (the intron interrupts the first and second bases of the codon) for introns 3, 4, and 6; and type 2 (the intron interrupts the second and third codon) for introns 8 and 12–14.Table IIExon/Intron organization of RGS6 geneTable IIExon/Intron organization of RGS6 gene Splicing of Human RGS6 Transcripts—We examined the relationship between exon and intron locations of the RGS6 gene to the structure of the 36 RGS6 cDNA variants we identified to gain insight into how these unique RGS6 variants arise. Fig. 3 illustrates the deduced splicing mechanisms involved in generating these 36 RGS6 transcripts. Transcripts encoding the two N-terminal forms of RGS6 arise by use of different transcriptional start sites. Exons 1–7 encode the 5′-untranslated region and unique N-terminal domain of RGS6L proteins while an alternate transcription start site within intron 7 generates RGS6S transcripts whose 5′-untranslated sequence and translation start site are encoded within intron 7 (non-coding exons A, B, C, and D) and exon 8. The translational start site for RGS6S proteins begins at nucleotide 24 of exon 8, resulting in no reading" @default.
• W2005444914 created "2016-06-24" @default.
• W2005444914 creator A5007905274 @default.
• W2005444914 creator A5048166676 @default.
• W2005444914 creator A5080153002 @default.
• W2005444914 date "2003-08-01" @default.
• W2005444914 modified "2023-09-27" @default.
• W2005444914 title "Human RGS6 Gene Structure, Complex Alternative Splicing, and Role of N Terminus and G Protein γ-Subunit-like (GGL) Domain in Subcellular Localization of RGS6 Splice Variants" @default.
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